In vitro model for priocidal activity

ABSTRACT

A proteinaceous material has been found to show similar activity and treatment response to that of disease causing prions, such as CJD. A prion model which incorporates the proteinaceous material has a variety of applications. The model has an ability to be cultured either in vivo or in vitro, allowing rapid screening of potential drugs for treating animals or humans, or methods of treating food products or items which may come into contact with prions, such as medical or dental devices. Several treatment methods and materials have been developed using the model.

This Application claims the Priority of U.S. Provisional ApplicationSer. No. 60/327,460, filed Oct. 5, 2001.

BACKGROUND OF THE INVENTION

The present invention relates to the field of infectious diseases. Itfinds particular application as a method of evaluating the response ofPrions (Proteinaceous Infectious Agents) to various treatments, and willbe described with particular reference thereto. It should beappreciated, however, that the invention is also applicable to otherstudies of prion activity.

The term “Prion” is used to describe proteinaceous-infectious agentsthat cause relatively similar brain diseases in humans and/or inanimals, which are invariably fatal. These diseases are generallyreferred to as transmissible spongiform encephalopathies (TSEs). TSEsinclude Creutzfeldt-Jakob disease (CJD) and variant CJD (vCJD) inhumans, Bovine Spongiform Encephalopathy (BSE) in cattle, also know as“Mad Cow Disease,” Scrapie in sheep, and Wasting Disease in elk. All ofthese diseases attack the neurological organs of the animal or animalswhich are susceptible to the particular disease. They are characterizedby initially long incubation times followed by a short period ofneurological symptoms, including dementia and loss of coordination, andeventually death.

The infectious agent responsible for these diseases is thought to be asimple protein, with no associated nucleic acids. The pathogenicmechanism for such prion diseases is proposed to involve an initiallynormal host encoded protein. The protein undergoes a conformationalchange to an abnormal form (a prion), which has the ability ofself-propagation. The exact cause of this change is, at present,unknown. The abnormal form of the protein is not broken down effectivelyin the body and its accumulation in certain tissues (in particularneural tissue) eventually causes tissue damage, such as cell death. Oncesignificant neural tissue damage has occurred, the clinical signs areobserved.

Prion diseases may thus be classified as protein aggregation diseases,which also include several other fatal diseases, such as Alzheimer'sdisease and amyloidosis. In the case of CJD, the most prevalent priondisease in humans (occurring in roughly 1:1,000,000 of the population),about 85% of cases are thought to arise sporadically, about 10% arethought to be inherited, and about 5% arise iatrogenically.

There are currently no known effective treatments for prion diseases inanimals or humans, and death thus follows the onset of neurologicalsymptoms. Progress in the identification of target treatment drugs hasbeen slow, due to the inability to perform testing in vitro. To date, nomethods for culturing prions in media in the laboratory have beendeveloped. In vivo studies involve inoculating a test animal with prionsand examining the animal's response to a proposed treatment regime.Because progress of the disease is slow, these in vivo studies areinevitably lengthy and are thus not readily amenable to the screening oflarge numbers of potential drugs. In vivo mouse or hamster models havebeen engineered to be more susceptible to prions and are generally usedfor evaluations. In addition, because these diseases tend to be animalspecific, it is not known whether tests done on animals can be readilyapplied to humans.

Some research groups have suggested using a yeast prion model for drugevaluation and there has been some reports of an in vitro model to studyprion folding. However, there have been no studies which haveestablished correlations between the behavior of these proposed modelsand prion activity.

In the early 1980's, a novel replicating agent was isolated from thehuman intestinal tract. (Burdon, J. Med. Micro., 29: 145-157 (1989)).This agent was isolated from the ileostomy fluid (filtered through a0.2μ filter) of two patients with Crohn's disease, and could be culturedin vitro. It was given the name Ileal Fluid Dependent Organism (IFDO),although it has been subsequently found to survive in other media, suchas in the presence of pancreatin. Discrete brown colonies were observedon a specific, select growth media. On examination of this agent, it didnot appear to be viral, bacterial, or fungal in nature, but did appearto grow logarithmically and have unusual resistance to a variety ofantibiotics, and physical and chemical agents. The agent was also foundto have a high resistance to moist heat. This agent has not previouslybeen directly linked to prions or used in prion research.

Although prion diseases have not generally been considered to be highlycontagious, they can be transmitted within a species and, under certainconditions, from one species to another. It has recently been shown thatprion diseases may be transmitted via high risk tissues, including thebrain, spinal cord, and eye. Iatrogenic transmission has also beenreported, including transmission via dura mater grafting, cornealtransplants, pericardial homografts, human gonadotropin, and humangrowth hormone contamination. Transmission via medical devices has alsobeen reported, including through reuse of neurosurgical instruments,depth electrodes, and other devices used during surgeries in closeproximity to the central nervous system.

There is currently much speculation about the efficacy of conventionaldecontamination and sterilization methods for destruction of prions.Prions are notoriously very hardy and demonstrate resistance to routinemethods of decontamination and sterilization. Some recommended methodsinclude incineration, prolonged steam autoclaving, sodium hydroxide andsodium hypochlorite treatments at high concentrations (e.g., 1M NaOH orNaHClO₃ at 2% available Cl for 1 hr.). These aggressive treatments areoften incompatible with medical devices, particularly flexibleendoscopes and other devices with plastic, brass, or aluminum parts.Many devices are damaged by exposure to high temperatures. Chemicaltreatments, such as strong alkali, are damaging to medical devicematerials or surfaces in general. Glutaraldehyde, formaldehyde, hydrogenperoxide, most phenolics, alcohols, and processes such as dry heat,boiling, freezing, UV, ionizing, and microwave radiation have generallybeen reported to be ineffective. There is a clear need for products andprocesses that are effective against prions yet compatible withsurfaces.

One less aggressive treatment which has been investigated and shown tobe effective against prions is a peracetic acid formulation formulatedby STERIS Corporation, Mentor, Ohio, under the tradename STERIS 20™. Theformulation contains peracetic acid in a blend of buffers,anticorrosives, surfactants, and chelators, prepared in a use dilutionfor sterile processing at above room temperature.

However, there is currently no ready means of evaluating anti-prion(“priocidal”) treatments. Culturing prion-treated devices after proposedpriocidal treatments involves inoculating animals with washings from thedevices and observing the development of the disease if the priocidaltreatment is ineffective. This is a lengthy process and prone to errors,since the numbers of prions remaining on the devices may be relativelysmall. Additionally, there is a risk that prions which are not destroyedby the priocidal treatment may pose hazards to workers.

There are thus increased concerns among medical personnel regarding theproper care of patients identified as having prion diseases. There arealso concerns that the diseases may be transmitted, through reuse ofinstruments and the like, due to a failure to detect the disease stateprior to death of the infected patient. Additionally, the risksassociated with high, medium, and low risk tissues have not yet beenestablished. For example, tonsillectomy and dental procedures have beenconsidered to be low risk procedures for potential prion infection.However, recent evidence suggests the risks may higher, due to thefinding that prion infected tissues are being found outside the brain.It has also been suggested that there may be a link betweenprion-related diseases and similar disease states, such as Parkinson'sand Alzheimer's diseases.

The present invention provides a new and improved method for evaluationof priocidal activity, which overcomes the above-referenced problems,and others.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, a method forevaluating potential treatments for activity against prions orprion-related diseases is provided. The method includes subjecting aprion model to the treatment, the prion model being one which has beenshown to exhibit a response similar to that of prions to a treatmentdesigned to attack prions and evaluating the effect of the treatment onthe prion model as an indicator of the effect of the treatment on theprion or prion-related disease.

In accordance with another aspect of the present invention, a method oftreating an item which may be contaminated with prions is provided. Themethod includes treating the item with a composition which includes atleast one of nisin, manganese, and silver nitrate to reduce the level ofviable prions on the item.

In accordance with another aspect of the present invention, a method ofscreening proposed drugs for activity against prion related diseases orproposed treatment processes or chemicals for priocidal activity isprovided. The method includes exposing a prion model to the proposeddrug, chemical, or process and culturing any remaining prion model invitro, the prion model having been shown to exhibit similar responses toprions to a drug, chemical, or process.

In accordance with another aspect of the present invention, a method oftreating a subject having a prion related disease is provided. Themethod includes treating a sample contaminated with an IFDO with aproposed treatment agent, the IFDO having been shown to respond to othertreatment agents in a similar manner to the prion. If the treatmentagent is effective at attacking the IFDO, treating the subject with thetreatment agent in an effective amount.

One advantage of the present invention is that proposed prion diseasetreatments, pharmaceuticals, and priocidal agents can be screened invitro, without the need for extensive in vivo study.

Another advantage of the present invention is that proposed priondisease treatments and priocidal agents can be evaluated rapidly.

Another advantage of the present invention is that prion-contaminatedinstruments, hard surfaces, and food products are rendered safer foruse.

Still further advantages of the present invention will become apparentto those of ordinary skill in the art upon reading and understanding thefollowing detailed description of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may take form in various components and arrangements ofcomponents, and in various steps and arrangements of steps. The drawingsare only for purposes of illustrating a preferred embodiment and are notto be construed as limiting the invention.

FIG. 1 is a schematic side view of a biological indicator containing aprion model according to the present invention;

FIG. 2 is a schematic side view of the biological indicator of FIG. 1after closing a cap to seal the indicator and mix the prion model withgrowth media;

FIG. 3 is a plot of prion model count against time for peracetic acidtreatment processes at different initial concentration levels ofperacetic acid;

FIG. 4 is a plot showing the total number of red blood cells over timefor a first control sample (water-RBC), a second control sample(pancreatin-Panc) and a sample of the prion model (labeled IFDO);

FIG. 5 is a plot of percent red blood cells having a normal structure(i.e., free of abnormal structure or aberrations) over time for a firstcontrol sample (water-RBC), a second control sample (pancreatin-Panc)and a sample of the prion model (labeled IFDO).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

An unusual proteinaceous entity which will be referred to herein as the“test protein” or “prion model” has been found to correlate with prionactivity and is thus useful as simulated prion model. The prion modelhas been developed as an indicator for prion inhibition/inactivation andhas shown correlation to prion inactivation in vitro and in vivo.

The preferred test protein was originally isolated by D. W. Burdon,Department of Microbiology, Queen Elizabeth Hospital, Birmingham,England. It was originally extracted from ileal fluid from patientssuffering from Crohn's disease and is proteinaceous in nature. Itappears to have associated iron and may have an RNA component.Microscopic examination at 100× shows the prion model to comprisepleomorphic particles of no uniform size or shape. We have subsequentlyshown this agent to be present in blood and serum samples.

It is contemplated, however, that other similar proteins, which show thesame or similar resistance to destructive treatments as the present testmodel, may alternatively be used as the prion model.

The test protein is readily cultured in an artificial medium, such asagar-based media or liquid media. It can be used in the medium orsuspended in water or other fluid. The protein appears to groweffectively when lysed red blood cells are present, thus it ispreferable that the media contain red blood cells from humans or otheranimals, or extracts therefrom, such as hemoglobin. Since the prionmodel has been found in a sizeable proportion of blood samples, it ispreferable for the blood to be screened prior to use in the growthmedium to avoid any competing reactions in vitro and contamination ofsamples later to be cultured in vivo.

Very few actives have to date been shown to be effective at inhibitingthe growth of the prion model in culture media. Of those found to havean effect, a number of these have previously been shown to have someactivity against prions including vCJD, showing the correlation of themodel with actual prion activity. These effective actives include sodiumhydroxide (about 1M NaOH) and a phenol-based formulation sold under thetradename LpH™ by STERIS Corporation, Mentor, Ohio. A peracetic-acidbased formulation, STERIS 20™, has previously been found to haveactivity against prions and has now been found to have activity againstthe prion model. Other actives with measurable activity, which have nowbeen identified using the prion model, include nisin, neomycin sulphate,silver nitrate, and manganese.

Because of the strong correlations which have been found between theresponse of prions to various treatments and the corresponding responseof the present test protein, the test protein can be used in vitro as amodel to screen large numbers of potential drugs, priocidal agents,processes, and the like (all generally referred to as “treatments”), fortheir potential effect against prions and prion diseases. Treatmentswhich prove to be effective under in vitro studies with the prion modelcan then proceed to in vivo testing, for example, by inoculating ananimal with a prion and subjecting the animal to a treatment regimen(e.g., in the case of a proposed drug for treating a prion disease), orby exposing a prion sample to a selected treatment (such as a proposedpriocidal agent for use on medical instruments) and then culturing theexposed sample in vivo, to determine whether the prions have beendestroyed by the selected treatment.

Using the prion model, conventional anti-microbial treatments (e.g.,peracetic acid, steam, or vapor hydrogen peroxide) can be evaluated fortheir effectiveness against prions. Modifications to the conventionalantimicrobial treatments can be made with a view to optimizing theantimicrobial treatment so that the treatment destroys or otherwiseinactivates prions and also destroys or otherwise inactivates othermicroorganisms typically destroyed by such processes. The modifiedprocess can then be used to treat devices contaminated with both prionsand with microorganisms. Novel actives, specifically designed fortreating prions, can also be evaluated.

Another use for the prion model is to develop treatments for foodproducts, generally those containing an animal product, which may becontaminated with prions. Such products include, for example, raw meat,including meat carcasses, ground meat, or chopped meat products, andcooked meat products, such as sausages, hams, processed meat productsand the like. Using the prion model, several treatments have beenidentified. A method of treating a food product for animal or humanconsumption which may be contaminated with prions has therefore beendeveloped. The method includes treating the food product with acomposition which includes at least one of nisin, manganese, and silvernitrate in a sufficient concentration and for a sufficient time toreduce the level of prions on the food product or to inactivate theprions present. Animal products which are rendered or otherwise preparedfor use as animal feedstocks may also be treated with the composition,either before or after rendering.

In another embodiment, a method of treating items, such as medical ordental instruments, which may be contaminated with prions has beendeveloped. The method includes treating the items with a compositionwhich includes at least one of nisin, manganese, and silver nitrate toreduce the level of prions on the items. The concentration of the activein the composition and the length of treatment will depend on the typeof active and degree of reduction sought. The treatment may be combinedwith other treatments known to reduce prions, such as steam autoclavingand/or sodium hydroxide or sodium hypochlorite treatment.

The composition for food or instrument treatment is preferably in liquidform, e.g., an aqueous solution of the active, although dry andnon-aqueous compositions are also contemplated. Treatment may include,for example, immersion of the items to be treated in the treatmentsolution, spraying or otherwise contacting the items with the solution,or exposing the items to a vapor containing the active.

Another treatment method which has been developed using the prion modelis to treat items which may have been contaminated with prions withoxidizing agent-based gaseous or liquid processing. As an example, aperacetic acid based solution is used. The method is useful fortreatment of medical and dental instruments, and the like, particularlythose which have been used for brain or related surgeries. Temperaturehas been found to have a significant effect on priocidal activity. Theperacetic acid solution is preferably at a temperature of from 45-60°C., more preferably, from 53-57° C. It is postulated that a reduction inactivity at higher temperatures may be due to coagulation of theprotein, rendering it less accessible to the peracetic acid. Thetemperature range is also effective for destruction of othercontaminants, such as microorganisms. This allows the same process to beused for both sterilization and prion reduction or elimination. Theperacetic acid solution preferably contains buffers, surfactants,chelants, and may also contain anticorrosives for reducing damage to theinstruments or to the treatment system. Surfactants are thought to beparticularly important in the formulation, since they may affect theconformational structure of the prion model protein and allow theperacetic acid to be more effective. A peracetic acid concentration ofat least 2000 ppm is preferred for rapid prion treatment, morepreferably, about 2500 ppm. Peracetic acid concentrations in the rangeof 2000 to 2500 ppm have been found to break down the protein to a formwhich is inactive (small peptides or amino acids). Lower temperaturesand concentrations are generally less effective. Higher temperatures maybe damaging to temperature-sensitive devices and also reduce the halflife of peracetic acid.

The peracetic acid treatment can be used to replace conventional,aggressive prion treatments such as steam/NaOH treatments. Or, theperacetic acid treatment can be used in combination with othertreatments. One proposed treatment regiment includes:

-   -   1. Cleaning the instruments with a cleaning agent which has been        found to be effective at removing proteinaceous material,        particularly prions;    -   2. Heat treatment with steam or high temperature (e.g., 180° C.)        thermal rinse (optional, or may be carried out after step 3.);    -   3. Sterilization treatment, e.g., with a peracetic acid        formulation at 2000 ppm, or above and a temperature of 55-57° C.        for 10-30 minutes.

The cleaning agent is preferably an alkaline cleaning agent or anenzymatic cleaning agent. Tests with the prion model have shown anincrease in the effectiveness of the treatment process with thealkalinity of the cleaning product. Highly alkaline products, such assodium or potassium hydroxide-based products, e.g., CIP™ 100 (obtainedfrom STERIS Corp., Mentor, Ohio) have been found to be particularlyeffective. Even though termed “highly alkaline,” these products have amuch lower alkali concentration (CIP™ 100 at 2 oz/gal contains 0.07 MKOH) than the 1N NaOH currently being recommended as a treatment forprions. Thus, a combination of an alkaline cleaning product wash at analkali concentration of about 0.02M to about 0.1M followed by aperacetic acid treatment is an effective alternative to a treatment with1N NaOH and is less damaging to the medical instruments or other itemsbeing treated.

In another embodiment, a method of screening proposed drugs for activityagainst prion related diseases or proposed treatment processes orchemicals for priocidal activity has been developed. The method includesexposing a prion model to the proposed drug, chemical, or process andculturing any remaining prion model in vitro. The prion model is onewhich has been shown to exhibit similar responses to prions to otheractives and processes.

In another embodiment, a method for evaluating the effect of a proposedtreatment, drug, or other active against prions involves exposing theprion model to the proposed treatment, drug, or other active. Afterexposure, the prion model (or whatever remains viable) is grown in asuitable growth medium.

For example, in the case of a treatment process, such as a sterilizingprocess, a coupon or instrument is contaminated with the prion model andexposed to a sterilizing or cleaning treatment. A swab, sample, orextraction is taken after the treatment and is placed in a growthmedium. If growth of the prion model is observed, the sterilizing orcleaning treatment has not been fully effective at destruction orremoval of prions.

In the case of an evaluation of a potential active, a solution of thedrug or other active is mixed with the prion model. After a selectedexposure time, an aliquot of the solution is taken and cultured in thegrowth medium. Prior to culturing, the aliquot is preferably neutralizedwith a suitable neutralizing agent to inactivate the drug or otheractive under test. If the drug or active proves effective against theprion model, it can be used in an effective amount to treat a subject,such as a contaminated surface or a person or animal suffering from aprion related disease.

In one embodiment, effects on the growth medium are used as an indicatorof residual prion model (and hence, by inference, of prion) activity.The growth medium preferably contains hemoglobin, either in a pure orrelatively pure form, or mixed with other blood related products. Forexample, the growth medium may contain lysed whole blood or lysed redblood cells. If any of the prion model has survived the treatmentprocess or exposure to the selected active, the hemoglobin content ofthe growth media is reduced as the prion model “grows.” This can beobserved by a reduction in the deep red color of the media, or by otherdetection techniques, such as chemical analysis, colorimetry,spectroscopy, or the like. If the media is a solid media, e.g., an agargel, the color change can be observed as a ring around the prion model,which increases in size over time. Thus, destruction/inactivation of themodel may be measured in term of the size of the hemoglobin ring at aparticular time after the start of the culturing process. Or, as withliquid media, a change in color of the media may be used as anindication of the amount of prion model remaining.

In one embodiment for testing a proposed treatment process (such as anoxidizing agent-based process, e.g., one using hydrogen peroxide or aperacid alone or in combination, in liquid or vapor form) for destroyingprions on medical instruments or other devices, a prion model iscontained in a biological indicator of the type conventionally used totest a sterilization or disinfection process for activity againstselected, usually relatively hard to kill, microorganisms. One suchindicator is exemplified in FIG. 1, although other known biologicalindicator designs may alternatively be used. The indicator includes avessel 10, which contains a sample 12 of the prion model, preferablycontained within the vessel such that it is not readily washed orotherwise removed from the vessel during the treatment process. Theexact mode of containment will depend on the type of treatment process.In the case of liquid sterilants, such as peracetic acid, the prionmodel is contained within a protein impermeable barrier, which ispermeable to the liquid sterilant. For gaseous sterilants, such as vaporhydrogen peroxide, or vapor peracetic acid, the prion model may besimply deposited on an interior surface 14 of a wall of the container orsupported on a carrier 16, such as a disk of paper, stainless steel, orpolyflex. A suitable growth media may be added to the indicator justbefore subjecting the indicator to the proposed treatment process. Morepreferably, the prion model is mixed with a suitable growth media aftertesting. In one embodiment, a growth media 18 is contained in apenetrable portion or frangible ampule 20 within the vessel and thenmixed with the prion model after removal of the indicator form thetreatment process. One way to mix the growth medium and the prion modelis to break the ampule or penetrate a wall 22 of the portion by movementof a cap 24 from an open position, shown in FIG. 1, in which thesterilant has access to the vessel through openings 26, to a closedposition, shown in FIG. 2, in which the cap seals the vessel, preventingexit and entry of fluids. In the embodiment of FIGS. 1 and 2, the caphas a projection 28 which pierces the wall 22. Growth of remaining prionmodel is observed, for example, by a color change of the media or otherdetectable change in a physical or chemical property of the media. Thecolor change may be due, for example, to production of the prion model(which appears blackish), reduction in the hemoglobin content of themedia, or may be a result of an interaction between the prion model anda chemical indicator present in the media. An observed change in coloris indicative that the proposed treatment process would not beeneffective as a treatment process for prion contaminated devices or otheritems.

In one embodiment, the indicator contains both a sample of the prionmodel and a sample of a microorganism which is known to have a highresistance to the type of treatment process under investigation. Aftersubjecting the indicator to the proposed treatment process, theindicator is evaluated for residual prions and/or microorganisms.Treatment processes which are effective against both prions andmicroorganisms can thus be developed or optimized.

Growth Media

A suitable growth media has been developed for culturing the prionmodel. The growth media contains an agar or broth base, such asMycoplasma™ agar or broth base, obtained from Oxoid. A source ofhemoglobin, such as washed and lysed red blood cells from a human orother animal, is also present. As discussed above, the red blood cellsare preferably tested to make sure that no prions are present in thegrowth media prior to use. The media also preferably contains a sourceof proteases. The source of proteases may be an isolated protease, anenzyme extract, which may also contain other enzymes, such as lipases,or a comminuted animal tissue, such as pancreatin, which is obtained byhomogenizing pancreas tissue. A dispersing agent, such as Tween 80, ispreferably present. Thallium acetate is an optional component forreducing media contamination. The ingredients are blended with water,preferably distilled or other purified water.

The following exemplary growth media has been developed for use with thetest protein: Distilled water 1000 mL agar or broth base 10-100 g (e.g.,Oxoid Mycoplasma ™) Dispersant (e.g., Tween 80) 0-5 mL Washed and lysedhorse red blood cells 10-40 mL Horse serum 0-10 mL 0.1 g/mL Pancreatin10-40 mL 2% thallium acetate 2-20 mL

To test the “viability” of the prion model (e.g., after a proposedtreatment process), an aliquot of a culture or dilution containing theprion model is inoculated into a known volume of the above culture mediaand incubated at around 37° C. for about 48-72 hours. The test proteinswill “grow” as a black precipitate in the liquid culture or as discrete‘colonies’ under the surface of a culture media plate. Although theprion model is not an organism, as is conventionally understood, theprion model does increase in amount on culturing and hence the term“grow,” and similar terms, are used herein to denote an increase in theprion model. The term “viability” is used to denote prion model capableof exhibiting growth, i.e, it has not been destroyed or otherwiseinactivated.

To prepare for in vitro tests, liquid cultures of the test protein arerapidly spun down at about 5000×g for about 5 minutes and washed inwater or fresh media.

Testing of the simulated priocidal activity can be performed similar totypical tests with bacteria or fungi. These methods include:

-   -   1) Minimum inhibitory concentrations (MIC): this method allows        for the rapid evaluation of a wide variety of actives to inhibit        the culturing of the test protein. In a simple set-up with a        microwell plate, serial dilutions of an active are performed and        a standard low concentration (e.g., <10⁵ entity forming units        (efu's)) of the prion model in growth media are added to each        dilution. Following incubation at 37° C. for 48-72 hours, the        presence of growth is indicated by a black precipitate at the        base of a well and the lowest concentration of an active to        inhibit the growth is recorded as the MIC. This method may be        used to identify possible drug targets or biocides for        anti-prion activity.    -   2) Time kill experiments: this method allows for the        determination of active or formulation efficacy over time. A        test material (e.g., a liquid formulation, product, or active)        is prepared at a variety of concentrations and under a variety        of environmental conditions (e.g., pH, temperature, presence of        a soil, and the like). The prion model culture, at a known        concentration, is then directly added to the test liquid and        aliquots removed over time for evaluation. The aliquots are        preferably neutralized to inhibit further activity of the test        material. Evaluation may include serial diluted of the aliquot        and plating on selective media to determine the reduction of the        test protein over time under the selected test conditions.        -   In another test, the culture containing the test protein may            be inoculated onto a substrate and exposed to a liquid or            gas phase active for selected time exposures. Recoverable            test protein is determined by serial dilution and plating.    -   3) Protein Degradation Studies. Protein containing media (either        the test protein or another protein such as one having a high        proportion of β sheet structure, similar to prion protein) are        subjected to a treatment procedure, such as a peracetic acid        treatment, and then aliquots are evaluated by gel        electrophoresis or other technique capable of separating out        complete proteins from smaller fragments. Several effective        priocidal agents have been found to break down the prion protein        into smaller fragments. Thus, agents which break down the test        protein or other proteins can be viewed as potential        prion-treating agents.

Such tests are valuable for optimizing the effectiveness of a givenformulation, active, or process against the test protein. This isparticularly important in understanding the effects of formulation andenvironmental factors on the activity of actives/biocides againstprions. Preferably, more than one test is carried out, for example, atime kill study and a protein degradation study are carried out for theproposed treatment before proceeding to evaluate the proposed treatmenton prions themselves.

The following examples indicate the effectiveness of the prion model asa model for actual prions and on the effectiveness of various proposedpriocidal treatments.

EXAMPLES Example 1 Growth Media

The following growth media was prepared: Distilled water 1000 mL OxoidMycoplasma ™ agar or broth base 35.5 g Tween 80 2 mL Washed and lysedhorse red blood cells 20 mL Horse serum 1.4 mL 0.1 g/mL Pancreatin 20 mL2% thallium acetate 7 mL

When the test protein is inoculated on to a supplemented mycoplasmabroth base plated media dish with the formula listed above, and culturedat 37° C., the inoculated spots yield discrete brown colonies on after48 hours, under aerobic, microaerophilic or anaerobic conditions. Nogrowth is observed at 4° C.

Example 2 Composition of the Test Protein

The test protein was analyzed and determined to contain amino acids, asnoted below, and at least two peptides.

When subjected to ICP, primarily iron was observed (although backgroundcalcium was seen, presumably all from the media).

Total Amino Acid Analysis

Cultures of the prion model were growth in broth, vigorous washed (byvortexing in saline 5 times) and dried. Samples were submitted for totalamino acid analysis. Samples were hydrolyzed for 26 hours in 6N HCl at110° C., dissolved in 0.01N HCl and analyzed by chromatography.

The results showed the presence of amino acids, with the followingpercentages: ASP 8.30% THR 3.36% SER 9.08% GLU 7.38% PRO 5.24% GLY10.96%  ALA 7.11% VAL 5.97% ILE 2.41% LEU 12.14%  TYR 1.61% PHE 3.60%LYS 5.84% HIS 11.26%  ARG 5.70%Protein Analysis

The prion model protein was solubilized and protein gels were run on thesupernatant. SDS-PAGE was used to separate the protein. The presence ofa diffuse protein band was observed above the dye front (<10 kDa). Thisband was transferred onto a membrane by Western blotting and submittedto CCF, Molecular Biology Core for N-terminal sequencing analysis. Thesignal was weak but indicated two peptide sequences as follows:KLL/DH/WQSQ/LHK/MQRF IQKHILQK/IM/LALE

Example 3 Correlation Studies

To demonstrate the effectiveness of time-kill tests, and to establish acorrelation between the response of the test protein with that of knownprions, the log reduction of test protein was determined using activesknown to be effective against prions. Log reduction is the differencebetween the log of the original number of organisms present (in thiscase, the number of test proteins, or, alternatively, the concentrationof test protein in the sample) and the log of the number remaining. Goodcorrelations were found for such actives. Examples are given below:

a) Peracetic Acid Studies

Specific Peracetic Acid formulations (STERIS 20™ obtained from STERISCorp., Mentor Ohio) previously proposed as effective priocidal agentswere found to be effective against the prion model. STERIS 20™ is aperacetic acid-based sterilant containing buffers, surfactants,chelating agents, and anticorrosives. The results of these tests showthat the temperature of the formulation is an important factor.Temperature and active concentration were found to have a surprising andsignificant effect on both protein and prion inactivation.

i) Protein Degradation Studies

Protein degradation studies were carried out by exposing samples of theprion model to a sterilant (such as peracetic acid) and to controlsolutions (e.g., water or tris-buffered saline, TBS). After exposure,the peracetic acid was neutralized immediately with sodium thiosulfate(STS) and the sample is separated by gel electrophoresis. Sodium decylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is effective as aprotein separating medium. Further, HLC analysis has shown that a majorcomponent of the model is a short peptide structure similar to thesimple amino acid structure of collagen. The separated proteins weretransferred to nitrocellulose by the Western blot method. The presenceof particular proteins, such as the prion model, was detected byimmunoblotting with monoclonal antibodies specific to the protein andquantified by the percentage intensity of immunostained proteins.Peracetic acid, when used in the particular formulations tested (STERIS20™), reduced the amount of whole protein, creating smaller peptides,which are regarded as inactivated for purposes of the prion model.

ii) Plating Studies

The efficacy of the peracetic acid formulations under differenttemperatures and concentrations was studied by suspension testing, usingthe prion model. Peracetic acid formulations were prepared at 1000,1500, and 2000 mg/L peracetic acid and maintained at about 50° C. Analiquot of the test protein suspension was directly added to eachformulation and samples removed and quantified by serial dilution andplating into modified Mycoplasma agar.

Time kill experiments were carried out to show the effect of time andperacetic acid concentration on the prion model. As an example, theeffect of Peracetic acid concentration at 50° C. was shown to besignificant (FIG. 3). Following incubation at 37° C. for 48 hours, theplates were counted and log reductions determined. FIG. 3 shows theeffect of Peracetic acid concentration (in mg/L) at 50° C. for 12 minsin the STERIS 20™ formulation. At 2000 mg/liter, a log reduction from 9log (initial concentration) to 2 logs was observed after 6 minutes.

For these tests, the peracetic acid was combined with the bufferingsystem used in the STERIS 20™ formulation, the only differences being inthe amount of peracetic acid employed.

These results are similar to a reported reduction of vCJD on Westernblots with the same STERIS 20™ peracetic acid formulation (Antloga, etal., Prion Diseases and Medical Devices, ASAIO J., S69-S72 (2000). Theseresults are being confirmed in vivo.

b) Phenolic Formulation Studies

LpH™ is a phenolic formulation sold by STERIS Corporation, Mentor, Ohio.The composition has previously been described as being effective againstscrapie in an in vivo study (Ernst & Race, Comparative Analysis ofScrapie Agent Inactivation Methods, J. Virological Methods, 41, pp.193-202 (1993)). The authors described the loss of scrapie infectivitydependent on the concentration and time of exposure. For example, at aconcentration of 0.9%, the infectivity removed, as measured by logreduction, was 5 logs after 0.5 hours and over 7 logs at 16 hours.Similarly, at 9%, an over 7 log reduction was observed at 0.5 hours.

The activity of the LpH™ product was tested on samples of the testprotein at a 5% LpH™ concentration. The results indicated a 5.2 logreduction at 1 hour and a 5.8 log reduction at 3 hours. Further, theproduct was shown to be more effective than a similar product LpHse™.The same differences between LpH™ and LpHse™ which has also been shownby Race for prions (unpublished results).

Example 4 Optimization of Peracetic Acid Treatment of Prion ModelSamples

Testing using protein degradation as a screening tool has shown that aperacetic acid concentration of about 2000 mg/L or above is preferredfor efficacy against the test protein (FIG. 3). These tests were carriedout using STERIS 20™.

The effect of temperature on the test protein formulation (studied at1000 mg/L peracetic acid) was found to be dramatic. The resultspreliminarily indicate that the peracetic acid formulation is highlyeffective at breaking down protein within the range of 50-57° C. at thetimes tested in this study. Little to no breakdown was observed attemperatures below 50° C. At about 60° C., or above, similar loss inactivity was observed. A temperature of about 55-57° C. has been foundparticularly effective.

Example 5 MIC Investigations

The effects of a variety of actives (many with previous reports ofpossible effects on prions) in MIC (growth inhibition) tests on theprion model were studied.

The following actives showed at least some effect on growthcharacteristics of the prion model (i.e., a reduction in the growth ofthe prion model):

-   -   nisin (^(˜)1000 mg/L)    -   Klenzyme™ (5%) (obtained from STERIS Corp.)    -   Renuklenz™ (5%)(obtained from STERIS Corp.)    -   NaOH (^(˜)0.01N)    -   HCl (0.1N)    -   Peracetic acid (^(˜)600 mg/L)    -   neomycin sulphate (^(˜)125 mg/L),    -   LpH™ (Obtained from STERIS Corp, Mentor, Ohio) (1-5%)    -   LpHse™ (Obtained from STERIS Corp, Mentor, Ohio) (1-5%)    -   Manganese (^(˜)100 mg/L)    -   silver nitrate (^(˜)30 mg/L)

Example 6 Removal Tests

A variety of cleaning agents were evaluated. Instruments werecontaminated with Bovine Serum Albumin (BSA-a protein). To make theprotein more difficult to remove, the instruments were heated at 110° C.for one hour to denature the protein. The instruments were then washedin an automated washer using 1 oz./gal. of a cleaning agent and a highwash temperature (150° C.). After the washing cycle, visual examinationfor remaining soil was carried out. The cleaning agents evaluated arelisted below in order of decreasing effectiveness. All the cleaningproducts were obtained from STERIS Corp., Mentor, Ohio.

-   -   CIP 100™ (a sodium hydroxide-based cleaner)—Most effective    -   CIP 150™ (a potassium hydroxide-based cleaner)    -   Process Klenz™    -   Criti-Klenz™    -   Renu-Klenz™ (a neutral product)    -   CIP 220™ (an acid-based cleaner)    -   Water—least effective

The above order of effectivity also generally follows (with theexception of water) the alkalinity of the product. The most effectivecleaning product, CIP 100™, also has the highest alkalinity. The leasteffective, CIP 220™, is acidic.

The same order of effectivity was found when the prion model was used inplace of BSA.

Example 7 Red Blood Cells Viability Assay

The prion model was found to adsorb/use hemoglobin or Red blood cells(RBCs) components in the growth media. A study of the effect of theprion model on whole red blood cells was carried out. RBCs were washedin saline 3 times and resuspended at a concentration to count cellsunder a Petroff Hauser at 40× (^(˜)100/field). Total red blood cells andthe percentage of cells which were still intact (unchanged inmorphology) were monitored over time following incubation with the prionmodel (IFDO) or with water (RBC) or pancreatin (Panc—this had been shownin preliminary experiments to lyse RBCs).

The results, shown in FIGS. 4 and 5, indicate that the prion modelcaused cell damage and lysis over time, when compared to the controls(RBC and Panc). There appears to be a lag time before red blood cellsare lysed by the prion model.

Example 8 Time Kill Experiments

Preliminary time kill experiments were conducted with the prion model.Results are expressed as log numbers (log₁₀), the lower the log number,the fewer the prions remaining and thus the more effective thetreatment.

Time kill experiments were carried out to show the effect of autoclavingthe prion model at 121° C. in saline solution. The prion count droppedfrom 8.0 logs initially to 5.5 logs after 15 minutes.

Preliminary studies in a gravity drain cycle for 1 hour at 134° C.showed the treatment was effective.

Time kill experiments were carried out to show the effect of ethyleneoxide on coupons treated with the prion model. Coupons exposed at 600mg/L for 18 mins. Prion count in Logs Initial count 8.5 Exposure at 70°C., 100% RH 5.0 Exposure at 54° C., 40% RH 4.7

As with peracetic acid, similar temperature effects were found, theethylene oxide being more effective at 54 than at 70° C.

Liquid Disinfectants/Sterilants were evaluated in time kill studies fortheir effectiveness against the prion model. All of the products wereobtained from STERIS Corp. Samples of the prion model in liquidsuspension were directly inoculated into each product. The results areshown below. Time of Log No. of Treatment Exposure (hrs) Prion ModelInitial count 0 ˜6.6 10% CIP220 ™ 1 >3.0 10% CIP220 ™ 3 >3.0 LpH ™ 1 1.4LpH ™ 3 0.8

Example 9 Investigation of Intrinsic Contamination

Blood products were screened for contamination with prions. Thefollowing levels of contamination were found:

Horse blood (1 lot) contaminated

Horse serum (1 lot) contaminated

Bovine serum (3 lots) one lot contaminated

Sheep's blood (2 lots) none contaminated

The results indicate that prion contamination of blood products iscommon and therefore all blood products to be used in prion-related workshould be screened for prions prior to use.

The invention has been described with reference to the preferredembodiment. Obviously, modifications and alterations will occur toothers upon reading and understanding the preceding detaileddescription. It is intended that the invention be construed as includingall such modifications and alterations insofar as they come within thescope of the appended claims or the equivalents thereof.

1-11. (canceled)
 12. A method of treating an item which may becontaminated with prions, the method comprising: treating the item witha composition which includes at least one of nisin, manganese, andsilver nitrate to reduce the level of viable prions on the item.
 13. Themethod of claim 12, wherein the item includes a food product for animalor human consumption.
 14. The method of claim 12, wherein the itemincludes a medical or dental device.
 15. The method of claim 12, whereinthe composition has been found to be effective at attacking an IFDO.16-19. (canceled)
 20. A method of treating a subject having a prionrelated disease comprising: treating a sample contaminated with an IFDOwith a proposed treatment agent, the IFDO having been shown to respondto other treatment agents in a similar manner to the prion; if thetreatment agent is effective at attacking the IFDO, treating the subjectwith the treatment agent.
 21. A method for treating an item for prioncontamination, the method comprising: exposing a prion model comprisingan ileal fluid dependent organism in vitro to one or more proposedchemicals or processes; culturing remaining prion model in vitroincluding growing the remaining prion model in a culture medium;evaluating the culture medium for activity of the chemical or processagainst prions; and treating the potentially prion contaminated itemwith one or more of the chemicals or processes which have been evaluatedfor activity against the prion model.
 22. The method according to claim21, wherein the treating step includes: applying one of the chemicalswhich has been evaluated as showing activity against the prion model toa surface of the item.
 23. The method according to claim 22, wherein theitem is an inanimate object.
 24. The method according to claim 22,wherein the item is an animate object and the chemical is applied to atissue surface.
 25. The method according to claim 24 wherein the tissuesurface includes a skin surface.
 26. The method according to claim 21wherein the culture medium includes hemoglobin and the step ofevaluating for activity against the prion model includes observing achange in color of the culture medium as an indication of the amount ofprion model remaining.
 27. The method according to claim 21, wherein thechemical includes a drug and further including: performing additionaltests for efficacy of the drug and safety to humans or animals.
 28. Amethod of treating an item which may be contaminated with prions, themethod comprising: treating the item with a composition that has beenevaluated to have activity against prions, which composition has beenevaluated by contacting a substrate to which a prion model has beenapplied with the composition and evaluating in vitro the activity of thecomposition as an indicator of activity against prions.
 29. The methodaccording to claim 28, wherein the prion model includes an ileal fluiddependent organism.
 30. The method according to claim 29, wherein thecomposition is an oxidizing agent.
 31. The method according to claim 29,wherein the evaluating step includes: culturing the ileal fluiddependent organism on the substrate in a growth medium; and, detectinggrowth of the ileal fluid dependent organism.
 32. The method accordingto claim 31, wherein detecting includes: subjecting the cultured ilealfluid dependent organism to a procedure which detects proteins.
 33. Themethod according to claim 28, wherein evaluating includes culturing theprion model in a media which contains hemoglobin and observing a changein color of the media as an indication of an amount of the prion modelemaining.
 34. The method according to claim 28, wherein treating theitem includes applying the candidate composition to a surface ofbiological tissue.